The goal of this project is to design small molecules which will inhibit the interaction between VEZF1 (vascular endothelial zinc finger 1) and DNA. Resulting small molecule drug candidates will be synthesized to provide material for in vitro screening. Previously, through standard recombinant DNA methods, VEZF1 expression vectors and VEZF1 responsive promoter-reporter genes were generated, the latter as cell-active luciferase reporters that quantitatively establish transcription activity. Using this sensitive cell-based assay, VEZF1/DNA candidate inhibitory compounds will be screened for their ability to selectively block VEZF1-mediated transcription as measured by the luciferase reporter assay. Control assays to counter screen for general toxicity or non-selective transcriptional inhibition will be employed in parallel. Zinc finger transcription factors do not have the typical, deep binding pockets often found in enzymes. DNA appears to bind to zinc finger transcription factors by forming interactions primarily with surface atoms. Using a homology model of VEZF1 and an active lead compound, M1012, potential binding sites on the VEZF1/DNA binding surface will be identified. Potential binding sites will have the following properties: 1) occupied by DNA upon formation of the VEZF1/DNA complex, 2) cavities with at least some hydrophobic regions, and 3) polar atoms available for hydrogen bonding with ligand atoms. Furthermore, an additional desirable feature would be for ligand interactions to occur with protein residues whose positions are well defined. Each potential binding site will be evaluated by using the FLO molecular modeling software to dock 40 compounds with experimentally determined activity in a luciferase reporter assay. The binding site model which is best able to distinguish between the active and inactive compounds will then be used for the design of new compounds. Using the predicted binding mode of M1012 (compound #1) and the de novo computer program AlleGrow, new compounds will be designed that 1) form interactions with the protein atoms similar to those formed by the lead compound and 2) display predicted affinities significantly greater than that calculated for M1012. Forty newly designed compounds which are synthetically accessible and have good predicted binding affinities will be submitted for synthesis and biological evaluation. This project which will use small organic molecules to probe the binding of VEZF1 to DNA to advance our general knowledge of zinc finger transcription factor DNA interactions. PUBLIC HEALTH RELEVANCE: The target of this study proposal is the "zinc finger" transcription factor that regulates blood and lymphatic vascular development. Zinc finger transcription factors are a subset of transcription factors utilizing zinc at its core for activity. In particular, we are targeting the zinc finger transcription factor, VEZF1 (vascular endothelial zinc finger 1). VEZF1 is essential for embryonic blood vessel formation and regulates the synthesis of important growth factors such as IL3, VEGFR1, VEGFR2, endothelin-1, neuropilin-1 and strathmin/OP18. We have undertaken a novel approach to design inhibitors of VEZF1/DNA binding using homology structural modeling and in silico targeting of small molecules to the VEZF1/DNA interface. Previous modeling work undertaken by VasculoMedics has identified a first generation series of small molecule antagonists which show activity in in vitro assay of VEZF1, a VEZF1 responsive promoter-reporter gene cell-active luciferase assay quantitatively establishing VEZF1 transcription activity and in vivo by inhibiting angiogenesis in the murine oxygen-induced retinopathy model. Since transcription factors are functionally closer to the ultimate pathological protein(s), specific inhibition of transcription factors may result in a greater degree of disease fighting activity along with reduced level of toxicity. This may have advantage over conventional small molecule drugs that directly inhibit their target protein through a one-to-one interaction as transcription factor inhibitors will literally turn off pathological protein manufacturing capability at its source in pathological conditions such as cancer and retinal diseases of neovasculature. Disruption of the interaction between zinc finger transcription factors and DNA is a method to activate or repress gene expression. Our project will explore VEZF1/DNA interactions using small organic molecules as probes. Our preliminary work in identifying first generation drug inhibitors of the VEZF1/DNA interaction is to our understanding first-in-kind in that neither small molecule drugs have been identified to inhibit VEZF1/DNA binding nor have small molecule drugs been described to inhibit zinc finger protein/DNA interactions. Work from this project will further advance our knowledge on zinc finger protein transcription factor/DNA interactions and may thus open the door to a whole new range of therapeutic agents.